Long-term effects of alendronate on fracture healing and bone remodeling of femoral shaft in ovariectomized rats

Abstract

Aim: To investigate the long-term effects of alendronate (Aln), a widely used oral bisphosphonate, on fracture healing and bone remodeling in ovariectomized rats.

Methods: Adult female SD rats underwent ovariectomy, and then bilateral femoral osteotomy at 12 weeks post-ovariectomy. From d 2 post-ovariectomy, the animals were divided into 3 groups, and treated with Aln (3 mg·kg(-1)·d(-1), po) for 28 weeks (Aln/Aln), Aln for 12 weeks and saline for 16 weeks (Aln/Saline) or saline for 28 weeks (Saline/Saline). At 6 and 16 weeks post-fracture, the fracture calluses were examined with X-ray radiography, and biomechanical testing and histological analysis were performed. The calluses were labeled with tetracycline and calcein to evaluate the mineral apposition rate (MAR).

Results: The fracture line was less distinct in the 2 Aln-treated groups at 6 weeks post-fracture, and disappeared in all the 3 groups at 16 weeks post-fracture. The size of the callus and radiographic density of the femora in the Aln/Aln group were the highest among the 3 groups at 6 and 16 weeks post-fracture. Similar results were observed in the ultimate load at failure and energy absorption. However, the treatment with Aln delayed endochondral ossification of the callus, and significantly increased the total sagittal-sectional area, percentage callus area and callus thickness, and decreased the MAR at 6 and 16 weeks post-fracture.

Conclusion: In the ovariectomized rat model, Aln is beneficial for the mechanical properties of the callus, but delays callus remodeling by suppressing the remodeling of woven bone into lamellar bone.

Figure 1

Experimental design showing the different treatment groups, time point of euthanasia, and agents used in different groups. The Aln/Aln group received Aln for 12 weeks before being subjected to a standardized fracture of the femora and during the fracture healing period for 6 and 16 weeks. The Aln/Saline group received Aln before the fracture and saline during the healing period. The Saline/Saline group received only saline during the experiment.

Figure 2

Soft X-ray radiographs of fractured femora. (A) The fracture line was visible in the control group at 6 weeks post-fracture, while it was invisible in all groups at 16 weeks post-fracture. (B and C) The relative density was quantitated and statistically analyzed (^cP<0.01 vs Saline/Saline).

Figure 3

Biomechanical properties of fractured calluses in the three treatment groups. The data are expressed as the mean±SD (^cP<0.01 vs Saline/Saline, n=6). (A) Ultimate load; (B) Energy absorption.

Figure 4

Histological changes in fracture healing at 6 and 16 weeks after osteotomy. At 6 weeks post-fracture, in the control group, mature woven bone (WB) had more integrity, and the callus was thick and continuous. In the Aln groups, the calluses primarily consisted of hyaline cartilage (HC). At 16 weeks post-fracture, the healing fracture callus remodeled better in the control group than the Aln groups. LB: lamellar bone, RC: remodeling callus. Scale bar represents 25 μm.

Figure 5

Quantitative analysis of the fracture calluses using HE staining (A, B, and C) and fluorescence labeling (D). The data are expressed as the mean±SD (^bP<0.05, ^cP<0.01 vs Saline/Saline, n=5). MAR: mineral apposition rate.

Figure 6

Representative fluorescence images of the fracture calluses at 16 weeks post-fracture.